Herpes simplex virus (HSV) provides a tractable genetic model system for eukaryotic DNA replication since it encodes at least 7 proteins which are involved directly in the synthesis of viral DNA, all of which are essential for virus replication. Two of these proteins are responsible for elongation of the DNA chain--the DNA polymerase (pol) catalytic core and its accessory protein UL42. These proteins form a stable complex in vitro and together perform processive DNA synthesis. The long term goal of the project is to understand the role of UL42 in DNA replication and how it interacts with pol for processive DNA replication. It is hypothesized that interference with the ability of UL42 to complex with pol will block viral DNA synthesis. Thus, identification of the domains of UL42 and pol which are critical for their interaction provides a target for the development of novel anti-viral compounds. Due to the structural conservation of pol accessory proteins among highly divergent species, it is possible that defining the structure of this complex will lead to an even broader approach for the rational design of compounds capable of inhibiting DNA synthesis in neoplastic cells. The specific aims of the project are 1) to determine the effect of UL42 on the mechanism of DNA polymerization using transient kinetic analysis; 2) to use the yeast two- hybrid system to identify the subdomains of UL42 and pol capable of physical interaction in vivo; 3) to select for non-interacting pol and UL42 mutants and extragenic suppressors in the yeast two-hybrid system following random mutagenesis using PCR or oligo-directed mutagenesis to alter charged residues; 4) to measure the affinities of wild-type and mutant proteins in vitro using GST-fusion proteins and to evaluate the relationship between these and the affinities demonstrated in the yeast two-hybrid system; 5) to construct HSV-1 and baculovirus recombinants and to analyze them for ori-dependent replication and as a means to screen for interference mutations; 6) to determine the hierarchy of protein:protein interactions during the assembly of proteins in the absence of DNA synthesis using immunofluorescence and confocal microscopy.